It is well accepted that stem cells possess the unique ability to self renew and differentiate into a diverse range of specialized cell types. For example, hematopoietic stem cells (HSCs) have provided an important window into stem cell biology as well as a valuable clinical tool for treatment of hematopoietic malignancies and other disorders. However, the complex signaling network regulating the balance between HSC self renewal and differentiation is still not well understood.
One important regulator of HSC homeostasis is suggested by the highly prevalent clinical finding that therapeutic lithium increases circulating HSCs (as CD34+ cells; Ballin, et al., 1998. Br. J. Haematol. 100:219-221) and peripheral blood counts (Boggs, et al., 1983, Semin. Hematol. 20:129-138; Joyce, 1984, Br. J. Haematol. 56:307-321; Ricci, et al. 1981, Haematologica. 66:627-633) in greater than 90% of patients taking lithium, and the laboratory findings that lithium also increases transplantable HSCs in mice (Boggs, et al., 1983. Semin. Hematol. 20:129-138; Joyce, 1984, Br. J. Haematol. 56:307-321). Because lithium directly inhibits glycogen synthase kinase-3 (GSK-3) (GSK-3; Klein, et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8455-8459), activating critical signaling pathways such as the Wnt and PI3K/PTEN/Akt pathways (Hedgepeth, et al., 1997, Dev. Biol. 185:82-91; Stambolic, et al., 1996, Curr. Biol. 6:1664-1668), these clinical and laboratory observations implicate GSK-3 as an important regulator of HSC homeostasis (Hedgepeth, et al., 1997, Dev. Biol. 185:82-91; Phiel, et al., 2001, Annu Rev. Pharmacol. Toxicol. 41:789-813; Focosi, et al., 2009, J. Leukoc. Biol. 85:20-28). Support for this hypothesis comes from pharmacological studies showing that HSCs and hematopoietic progenitor cells (HPCs) are increased, and hematopoietic repopulation is enhanced when BM transplant recipient mice are treated with alternative GSK-3 inhibitors (Trowbridge, et al., 2006. Nat. Med. 12:89-98; Goessling, et al., 2009, Cell. 136:1136-1147; Holmes, et al., 2008, Stem Cells. 26:1288-1297). Furthermore, mouse ES cells treated with GSK-3 inhibitors maintain pluripotency (Sato, et al., 2004, Nat. Med. 10:55-63; Ying, et al., 2008, Nature. 453:519-523), and mouse ES cells lacking Gsk3a and Gsk3b maintain expression of markers of pluripotency under conditions that induce control ES cells to differentiate (Doble, et al., 2007, Dev. Cell. 12:957-971). These observations suggest a negative role for GSK-3 in ESC renewal. However, Gsk-3 loss of function in HSCs has not previously been performed, and the downstream pathways regulated by GSK-3 in HSCs have not yet been established.
Canonical Wnt signaling, which inhibits GSK-3 and thereby stabilizes beta-catenin, plays a central role in the self renewal of diverse stem cell populations (Sato, et al., 2004, Nat. Med. 10:55-63; Doble, et al., 2007, Dev. Cell. 12:957-971; Staal, et al., 1999, Int. Immunol. 11:317-323; Reya, et al., 2005, Nature. 434:843-850; Staal, et al., 2008, Eur. J. Immunol. 38:1788-1794; Malhotra, et al., 2009, Cell Stem Cell. 4:27-36). A role for Wnt signaling in hematopoiesis is supported by observations that Wnt ligands enhance proliferation of HSCs ex vivo (Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, et al., 1998, Blood. 92:3189-3202; Willert, et al., 2003, Nature. 423:448-452) and that Wnt antagonists inhibit HSC proliferation and reconstitution (Reya, et al., 2003, Nature. 423:409-414; Jeannet, et al., 2008, Blood. 111:142-149). In addition, overexpression of stabilized β-catenin promotes HSC self renewal and proliferation ex vivo under certain conditions (Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, et al., 1998, Blood. 92:3189-3202; Willert, et al., 2003, Nature. 423:448-452; Reya, et al., 2003, Nature. 423:409-414; Baba, et al., 2006, J. Immunol. 177:2294-2303), and conditional deletion of β-catenin using vav-cre impairs HSC function in competitive repopulation assays (Zhao, et al., 2007, Cancer Cell. 12:528-541). Furthermore, long-term reconstituting capacity in serial transplants is impaired in HSCs recovered from fetal liver of Wnt3a KO embryos (Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, et al., 1998, Blood. 92:3189-3202; Willert, et al., 2003, Nature. 423:448-452; Reya, et al., 2003, Nature. 423:409-414; Baba, et al., 2006, J. Immunol. 177:2294-2303; Luis, et al., 2009, Blood. 113:546-554) or from adults overexpressing the Wnt inhibitor Dick in the hematopoietic niche (Fleming, et al., 2008, Cell Stem Cell. 2:274-283), which suggests that Wnt signaling is required to maintain the long-term repopulating activity of HSCs.
However, there are conflicting reports on the requirement for Wnt/β-catenin signaling in basal hematopoiesis: conditional disruption of β-catenin and γ-catenin/plakoglobin in adult HSCs does not affect their ability to self renew and reconstitute hematopoietic lineages (Jeannet, et al., 2008, Blood. 111:142-149; Cobas, et al., 2004, J. Exp. Med. 199:221-229; Koch, et al., 2008, Blood. 111:160-164). In addition, although overexpression of stabilized β-catenin increases immunophenotypic HSCs, this is associated with a loss of repopulating activity and hematopoietic failure in vivo (Scheller, et al., 2006, Nat. Immunol. 7:1037-1047; Kirstetter et al., 2006, Nat. Immunol. 7:1048-1056), findings that appear incompatible with a positive role for β-catenin in hematopoiesis. A general conclusion from these apparently conflicting reports is that the role of Wnt signaling in hematopoiesis is complex and context dependent (Staal, et al., 2008, Eur. J. Immunol. 38:1788-1794; Malhotra, et al., 2009, Cell Stem Cell. 4:27-36). However, although the β-catenin loss-of-function studies suggest that canonical Wnt signaling is not essential for basal hematopoiesis in adults, they do not rule out a possible role for the Wnt/β-catenin pathway under nonbasal conditions, and are still compatible with gain-of-function experiments in which the pathway is activated.
GSK-3 is also inhibited by Akt/PKB, which in turn requires the activity of PI3K and is antagonized by phosphatase and tensin homolog (PTEN), a PI3 phosphatase. Loss of Pten transiently increases HSCs, which is followed by progressive HSC depletion, increased lineage commitment resembling myeloproliferative disorder, and acute leukemia (Yilmaz, et al., 2006, Nature. 441:475-482; Zhang, et al., 2006, Nature. 441:518-522). This expansion and subsequent depletion in Pten KO HSCs is mediated through mammalian target of rapamycin (mTOR), as the phenotype is reversed by treatment with rapamycin (Yilmaz, et al. 2006, Nature. 441:475-482), and a similar HSC phenotype is observed with KO of tuberous sclerosis complex 1 (Tsc1), a negative regulator of mTOR (Chen, et al., 2008, J. Exp. Med. 205:2397-2408; Gan, et al., 2008, Proc. Natl. Acad. Sci. U.S.A. 105:19384-19389). As GSK-3 is an indirect target of PTEN and antagonizes mTOR through phosphorylation of Tsc2 (Inoki, et al., 2006, Cell. 126:955-968), inhibition of GSK-3 could mimic the hematopoietic phenotype of Pten and Tsc1 KOs.
Currently available pharmacological data from humans and mice suggest that GSK-3 is an important regulator of HSC homeostasis, but the pathways regulated by GSK-3 in HSCs have not been defined. Furthermore, Gsk-3 loss of function in HSCs/HPCs has not previously been reported for either Gsk3a or Gsk3b, and this is an essential step to defining the role of Gsk-3 within HSCs.
Without question, the maintenance and expansion of long-term transplantable HSCs under in vivo and ex vivo conditions is a crucial component and major challenge in stem cell research and therapeutic hematopoietic stem cell transplantation. Thus, the ability to maintain and expand HSCs in culture has long been deemed the holy grail of hematopoietic stem cell research. Unfortunately, the ability to expand HSCs consistently and effectively in ex vivo culture conditions has not yet been perfected. Therefore, there is a long felt need in the art for a culturing system and method for maintaining and expanding HSCs. By understanding the complex signaling network regulating the balance between HSC self renewal and differentiation, the present invention satisfies this need.